My research group has cloned a series of kinase-related sequences
using PCR amplification. We next used 3'Race (a procedure that
preferentially amplifes cDNAs related to a targeted primer sequence)
to obtain nearly the entire sequence of one class of the
kinase-related sequences. Our initial primers targeted a single class
of kinase (a disease resistance gene in plants). The question we
wished to address was whether this approach would select kinases
related to our gene of interest or would kinases in general be
amplified. To address this question we used SWISS-MODEL to develop
homology-based protein models of the disease resistance gene and the
amplified kinase sequences. We removed the six amino acids from the
amino terminal end of the disease resistance genes and all of the
domain XI before doing the comparisons. The six amino acids were
removed so the two sequences started at the same amino acid, and
domain XI was removed because it is the most variable region in
kinases. Therefore we were asking the question: how similar are the
predicted 3D structes of the two core sequences?
When we used SWISS-MODEL, the same five sequences were selected from
the protein data base for modeling purposes. When we compared these
to structures to the structure of alpha, cyclic AMP dependent protein
kinase, we noted that our two seqeunces had similar alpha helix and
beta chain changes relative to the other kinase. We next compared the
structures of these two proteins to 13 other plant protein kinases
which have diverse functions, but known related to disease resistance.
(Similar trucations were performed on these kinases.) Our two
sequences appeared to be unique from these other kinases. When the
models of the two sequences are compared we noted only a few changes.
The most notable change was in domain VIII, a domain shown by yeast
two-hybrid analysis to be critical for the fucntion of this disease
resistance protein.
I am in the process of writing a manuscript describing these
experiments and would like to know what those in the field of protein
modeling feel is the appropriate modeling information to present in
the manuscript. My field is plant molecular genetics, so I a new the
modeling field. So far our narrative describes the specific alpha
helix and beta sheet changes. Our figure shows where SWISS-MODEL
places these structures in the two sequences. What more can we add to
strengthen the manuscript.
I would like to thank any person who repsonds in advance. Hope this
is not too simple of a question. Feel free to respond here or via
e-mail.
Phil McClean
Dept of Plant Sciences
North Dakota State University
mcclean at plains.nodak.edu